Technical Abstract: The cauliflower (Brassica oleracea L. var. botrytis) Or allele confers the accumulation of high levels of beta-carotene in various tissues that normally are devoid of carotenoids. Early work revealed that the Or gene appeared not to exert its effect via direct up-regulation of genes that encode enzymes of the carotenoid biosynthetic pathway. To decipher the molecular basis of Or in regulating carotenoid accumulation in cauliflower, we have undertaken the isolation of Or by a map-based cloning strategy. Ten AFLP markers closely linked to Or were identified using bulked segregation analysis. Two markers flanking the locus were converted into locus-specific PCR-based SCAR markers. These markers were employed for the analysis of a segregating population consisting of 1632 F2 individuals and a high-resolution genetic linkage map of the Or region was developed. To facilitate positional cloning, we constructed a cauliflower BAC library using the Or homozygous plants. The library comprises 60,288 clones with an average insert size of 110 kb and represents approximately 10-fold coverage of the genome. A BAC contig encompassing the Or locus was established by screening the library with a marker closely linked to Or and by overlapping BAC clones through chromosome walking. Physical mapping delimited the Or locus to a 50-kb genomic fragment within a single BAC clone, which is currently being sequenced to identify candidate genes for genetic complementation. These results provide essential information for the isolation of this interesting and potentially important Or gene in regulating carotenoid accumulation.